Identification of a Ligand-binding Site in the Na+/Bile Acid Cotransporting Protein from Rabbit Ileum

Reabsorption of bile acids occurs in the terminal ileum by a Na+-dependent transport system composed of several subunits of the ileal bile acid transporter (IBAT) and the ileal lipid-binding protein. To identify the bile acid-binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7-azi-derivative of cholyltaurine followed by enrichment of IBAT protein by preparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypsin yielded IBAT peptide fragments in the molecular range of 20.4–4 kDa. With epitope-specific antibodies generated against the C terminus a peptide of molecular mass of 6.6–7 kDa was identified as the smallest peptide fragment carrying both the C terminus and the covalently attached radiolabeled bile acid derivative. This clearly indicates that the ileal Na+/bile acid cotransporting protein IBAT contains a bile acid-binding site within the C-terminal 56–67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the seventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site is localized to the ninth transmembrane domain and the C terminus.